Abstract
Repeated treatment of female rats with the synthetic estrogen ethynylestradiol (EE2) increases the formation of the cyclosporine A (CyA) metabolites AM1c and AM9 by 3-fold, whereas the formation of AM1 and AM4N is not significantly enhanced. The formation of all four CyA metabolites was inhibited by greater than 80% by the CYP3A-selective substrate midazolam or polyclonal anti-rat CYP3A IgGs in liver microsomes of untreated and EE2-induced rats. In contrast, anti-rat CYP2C6 IgGs had little effect, indicating the involvement of a CYP3A but not 2C6 in this EE2-stimulated CyA metabolism. Semiquantitative reverse-transcriptase polymerase chain reaction was used to determine the mRNA content for four CYP3A genes (CYP3A2, CYP3A9, CYP3A18, and CYP3A23) in livers of control and EE2-treated female rats. EE2 selectively induced CYP3A9 by 3.3-fold whereas the expression of CYP3A18 and CYP3A23 was slightly decreased; neither CYP3A2 mRNA nor CYP3A1 mRNA was detectable in these EE2-treated livers. To determine whether rat liver microsomal CYP3A9 was indeed responsible for the EE2-stimulated CyA metabolism, a recombinant CYP3A9 was heterologously expressed in Escherichia coli. When functionally reconstituted, this enzyme was active in metabolizing CyA preferentially to its AM9 and AM1c metabolites as compared with CYP3A4. These findings thus support the notion that the increased CyA-metabolizing capacity of EE2-treated female rat liver microsomes is due to the induction of the CYP3A9 enzyme.
Footnotes
-
Send reprint requests to: Walter Jäger, Ph.D., Institute of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria. E-mail:wjaeger{at}speedy.pch.univie.ac.at
-
This work was supported in part by National Institutes of Health Grants DK 26506 (M.A.C.), DA 04265 (L.M.B.), GM 26691 (L.Z.B.), and the Deutsche Forschungsgemeinschaft Ha 1124/6–1 (W.G.H.). A.M. thanks the Gottlieb Daimler- und Karl Benz-Stiftung for a scholarship. The authors also acknowledge the use of the University of California at San Francisco Liver Center Spectrophotometry facility supported by the National Institutes of Health Liver Core Center Grant NIDDK 26743.
-
↵2 In common with many other P-450 suicide inactivators such as secobarbital, allylisopropylacetamide, and cannabidiol, EE2, by virtue of also being an excellent lipophilic substrate, would inactivate the enzyme on acute administration, but on repeated administration would induce rat liver CYPs that may be different from that/those initially inactivated (Bornheim LM and Correia MA, 1989a,b; and references therein).
- Abbreviations used are::
- CyA
- cyclosporine
- EE2
- ethynylestradiol
- P-450
- cytochrome P-450
- RT
- reverse transcriptase
- PCR
- polymerase chain reaction
- DX
- dexamethasone
- GAPDH
- glyceraldehyde phosphate dehydrogenase
- DMSO
- dimethyl sulfoxide
- Received March 2, 1999.
- Accepted September 23, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|
